TBX18 overexpression enhances pacemaker function in a rat subsidiary atrial pacemaker model of sick sinus syndrome.

KEY POINTS: The sinoatrial node (SAN) is the primary pacemaker of the heart. SAN dysfunction, or 'sick sinus syndrome', can cause excessively slow heart rates and pauses, leading to exercise limitation and syncope, currently treated by implantation of an electronic pacemaker. 'Biopacemaking' utilises gene therapy to restore pacemaker activity by manipulating gene expression. Overexpressing the HCN pacemaker ion channel has been widely used with limited success. We utilised bradycardic rat subsidiary atrial pacemaker tissue to evaluate alternative gene targets: the Na+ /Ca2+ exchanger NCX1, and the transcription factors TBX3 and TBX18 known to be involved in SAN embryonic development. TBX18 overexpression restored normal SAN function, as assessed by increased rate, improved heart rate stability and restoration of isoprenaline response. TBX3 and NCX1 were not effective in accelerating the rate of subsidiary atrial pacemaker tissue. Gene therapy targeting TBX18 could therefore have the potential to restore pacemaker function in human sick sinus syndrome obviating electronic pacemakers. ABSTRACT: The sinoatrial node (SAN) is the primary pacemaker of the heart. Disease of the SAN, sick sinus syndrome, causes heart rate instability in the form of bradycardia and pauses, leading to exercise limitation and syncope. Biopacemaking aims to restore pacemaker activity by manipulating gene expression, and approaches utilising HCN channel overexpression have been widely used. We evaluated alternative gene targets for biopacemaking to restore normal SAN pacemaker physiology within bradycardic subsidiary atrial pacemaker (SAP) tissue, using the Na+ /Ca2+ exchanger NCX1, and the transcription factors TBX3 and TBX18. TBX18 expression in SAP tissue restored normal SAN function, as assessed by increased rate (SAN 267.5 ± 13.6 bpm, SAP 144.1 ± 8.6 bpm, SAP-TBX18 214.4 ± 14.4 bpm; P < 0.001), improved heart rate stability (standard deviation of RR intervals fell from 39.3 ± 7.2 ms to 6.9 ± 0.8 ms, P < 0.01; root mean square of successive differences of RR intervals fell from 41.7 ± 8.2 ms to 6.1 ± 1.2 ms, P < 0.01; standard deviation of points perpendicular to the line of identity of Poincaré plots (SD1) fell from 29.5 ± 5.8 ms to 7.9 ± 2.0 ms, P < 0.05) and restoration of isoprenaline response (increases in rates of SAN 65.5 ± 1.3%, SAP 28.4 ± 3.4% and SAP-TBX18 103.3 ± 10.2%; P < 0.001). These changes were driven by a TBX18-induced switch in the dominant HCN isoform in SAP tissue, with a significant upregulation of HCN2 (from 1.01 × 10-5  ± 2.2 × 10-6 to 2.8 × 10-5  ± 4.3 × 10-6 arbitrary units, P < 0.001). Biophysically detailed computer modelling incorporating isoform-specific HCN channel electrophysiology confirmed that the measured changes in HCN abundance could account for the observed changes in beating rates. TBX3 and NCX1 were not effective in accelerating the rate of SAP tissue.

Morphological characteristics of the sinus node on postmortem tissue.

The sinus node is an intensively researched structure in terms of anatomical, histological, electrophysiological, molecular and genetic approach. For postmortem diagnosis it is still difficult to investigate due to a still reduced accessibility. In this study we tried and succeed to apply molecular biology techniques on postmortem tissues in order to widen the range of postmortem forensic investigation and provide information related to the diagnostic of cardiac arrhythmia. We described the stages of this investigation, with dissection, preservation and analysis that included classical histology, immunohistochemistry, confocal microscope, microdissection, RIN testing, mRNA expression obtaining a precise morphofunctional location of the sinus node.

Structural remodelling of the sinoatrial node in obese old rats.

During ageing, the function of sinoatrial node (SAN), the pacemaker of the heart, declines, and the incidence of sick sinus syndrome increases markedly. The aim of the study was to investigate structural and functional remodelling of the SAN during ageing. Rats, 3 and 24 months old (equivalent to young adult and approximately 69-year-old humans), were studied. Extracellular potential recording from right atrial preparations showed that (as expected) the intrinsic heart rate was slower in the old animals. It also showed a shift of the leading pacemaker site towards the inferior vena cava in the old animals. Consistent with this, intracellular potential recording showed that slow pacemaker action potentials were more widespread and extended further towards the inferior vena cava in old animals. Immunohistochemistry demonstrated that SAN tissue expressing HCN4, but lacking the expression of Na(v)1.5 (lack of Na(v)1.5 explains why pacemaker action potential is slow), was also more widespread and extended further towards the inferior vena cava in the old animals. Immunolabelling of caveolin3 (expressed in cell membrane of cardiac myocytes) demonstrated that there was a hypertrophy of the SAN cells in the old animals. Histology, quantitative PCR, and immunohistochemistry revealed evidence of a substantial age-dependent remodelling of the extracellular matrix (e.g. approximately 79% downregulation of genes responsible for collagens 1 and 3 and approximately 52% downregulation of gene responsible for elastin). It is concluded that the age- (and/or obesity-) dependent decline in SAN function is associated with a structural remodelling of the SAN: an enlargement of the SAN, a hypertrophy of the SAN cells, and a remodelling of the extracellular matrix.

Inflammation-mediated retinal edema in the rabbit is inhibited by topical nepafenac.

The purpose of this study was to evaluate the ability of the nonsteroidal anti-inflammatory drug nepafenac to prevent development of mitogen-induced pan-retinal edema following topical ocular application in the rabbit. Anesthetized Dutch Belted rabbits were injected intravitreally (30 microg/20 microL) with the mitogen concanavalin A to induce posterior segment inflammation and thickening (edema) of the retina. The Heidelberg Retina Tomograph was used to generate edema maps using custom software. Blood-retinal barrier breakdown was assessed by determining the protein concentration in vitreous humor, whereas analysis of PGE2 in vitreous humor was performed by radioimmunoassay. Inhibition of concanavalin A-induced retinal edema was assessed 72 h after initiation of topical treatment with nepafenac (0.1-1.0%, w/v), dexamethasone (0.1%), VOLTAREN (0.1%), or ACULAR (0.5%). Concanavalin A elicited marked increases in vitreal protein and PGE2 synthesis at 72 h postinjection. Retinal thickness was also increased by 32%, concomitant with the inflammatory response. Topical application of 0.5% nepafenac produced 65% reduction in retinal edema which was correlated with 62% inhibition of blood-retinal barrier breakdown. In a subsequent study, 0.5% nepafenac significantly inhibited (46%) blood-retinal barrier breakdown concomitant with near total suppression of PGE2 synthesis (96%). Neither Voltaren nor Acular inhibited accumulation of these markers of inflammation in the vitreous when tested in parallel. This study demonstrates that nepafenac exhibits superior pharmacodynamic properties in the posterior segment following topical ocular dosing, suggesting a unique therapeutic potential for a variety of conditions associated with retinal edema.

A comparison of the effects of olopatadine and ketotifen on model membranes.

Olopatadine is a human conjunctival mast cell stabilizer with antihistaminic activity. Ketotifen is an older molecule that possesses antihistaminic activity and is reported to have additional pharmacological properties. The interactions of these two compounds with model membranes (i.e., monolayers of 1-stearoyl-2-oleoyl-sn-glycerophosphocholine at the argon-buffer interface), and natural (i.e., erythrocyte) membranes were compared in an effort to understand the differences in their biological activities. Drug-lipid interaction with monolayers was determined by monitoring the surface pressure as a function of the drug concentration in the aqueous phase supporting the monolayer. Drug interaction with erythrocyte membranes was determined by monitoring changes in the permeability of the membranes to hemoglobin and 6-carboxyfluorescein as a function of drug concentration in the medium. Olopatadine and ketotifen are both intrinsically surface active and both interact with phospholipid monolayers. However, in both the presence and absence of lipid monolayers, the changes in surface pressure induced by olopatadine are lower than those caused by ketotifen. The effects of these two drugs on cell membranes were dramatically different. Exposure of bovine erythrocytes to increasing concentrations of ketotifen (1-10 mM) resulted in complete hemolysis of the cells, whereas olopatadine (1-10 mM) caused only minimal hemolysis (< 8%). Consistent results were obtained in experiments measuring the leakage of 6-carboxyfluorescein from erythrocyte ghosts as a more sensitive marker of membrane perturbation. Olopatadine treatment (0.1-10 mM) minimally perturbed the cell membrane while ketotifen (1-10 mM) caused a concentration dependent release of the fluorescent marker. These data demonstrate fundamental differences between the two drugs in their effects on cell membranes. Moreover, the differences are consistent with the surface activities of the two compounds measured in monolayers and with reported differences in their pharmacological activities. These findings offer an explanation for the biphasic non-specific cytotoxic effect of ketotifen on histamine release from mast cells and may account for the nonlytic mast cell stabilizing activity of olopatadine.

Nepafenac, a unique nonsteroidal prodrug with potential utility in the treatment of trauma-induced ocular inflammation: I. Assessment of anti-inflammatory efficacy.

Nepafenac, the amide analog of 2-amino-3-benzoylbenzeneacetic acid (amfenac), was examined in preclinical models for its potential utility as a topical ocular anti-inflammatory agent. Diclofenac was selected as the reference compound. In contrast to diclofenac (IC50 = 0.12 microM), nepafenac exhibited only weak COX-1 inhibitory activity (IC50 = 64.3 microM). However, amfenac was a potent inhibitor of both COX-1 (IC50 = 0.25 microM) and COX-2 activity (IC50 = 0.15 microM). Ex vivo, a single topical ocular dose of nepafenac (0.1%) inhibited prostaglandin synthesis in the iris/ciliary body (85-95%) and the retina/choroid (55%). These levels of inhibition were sustained for 6 h in the iris/ciliary body and 4 h in the retina/choroid. Diclofenac (0.1%) suppressed iris/ciliary body prostaglandin synthesis (100%) for only 20 min, with 75% recovery observed within 6 h following topical dosing. Diclofenac's inhibition of prostaglandin synthesis in the retina/choroid was minimal. Nepafenac's inhibitory efficacy and longer duration of action was confirmed in a trauma-induced rabbit model of acute ocular inflammation monitoring protein or PGE2 accumulation in aqueous humor. Results warrant further assessment of nepafenac's topical ocular efficacy in the treatment of postoperative ocular pain, inflammation, and posterior segment edema.

Nepafenac, a unique nonsteroidal prodrug with potential utility in the treatment of trauma-induced ocular inflammation: II. In vitro bioactivation and permeation of external ocular barriers.

Nepafenac, the amide analog of the NSAID amfenac, was examined in vitro for its bioactivation by ocular tissue components and its ability to permeate external ocular barriers. Rabbit tissues catalyzed a concentration-dependent conversion of nepafenac to amfenac. The order of specific hydrolytic activity is retina/choroid >> iris/ciliary body. Corneal tissue showed only minimal activity. Similarly, in human ocular cadaver tissue the specific activity of iris/ciliary body was greater than cornea. Continued perfusion of the corneal epithelium demonstrated a nearly six-fold greater permeation coefficient for nepafenac (k(p) = 727 x 10(-6) min(-1)) than for diclofenac (k(p) = 127 x 10(-6) min(-1)). Superior permeation of conjunctival and scleral tissue by nepafenac (k(p) = 128 x 10(-6) min(-1)) compared to diclofenac (k(p) = 80 x 10(-6) min(-1)) was also evident. Short term perfusion (5 min) of the corneal surface with 0.1% nepafenac resulted in sustained flux of drug across the cornea for 6 h. Under identical conditions only 3.3 microM of diclofenac accumulated on the corneal endothelial side compared to 16.7 microM nepafenac. The enhanced permeability of nepafenac, combined with rapid bioactivation to amfenac by the iris/ciliary body and retina/choroid, make it a target specific NSAID for inhibiting prostaglandin formation in the anterior and posterior segments of the eye.

Angiostatic activity of steroids in the chick embryo CAM and rabbit cornea models of neovascularization.

Ocular neovascular diseases represent a major cause of blindness in the world. Angiostatic steroids are a unique class of compounds which inhibit the formation of new blood vessels in various models, including ocular models of angiogenesis. In search of potent new anti-angiogenic agents for the treatment of ocular neovascular disease, a large group of steroids were evaluated for angiostatic activity in the chick embryo CAM model. Angiostatic activity was found among all steroid classes included in the study. There was a good correlation between the angiostatic efficacies of 15 diverse steroids tested in the chick CAM and in the rabbit LPS-induced corneal pocket models of neovascularization (r=0.76, p=0.01). These studies show that potent angiostatic steroids inhibit neovascularization in two different animal models, suggesting a common mechanism of action. Glucocorticoid therapy is sometimes associated with ocular side effects. Two of the most potent angiostatic steroids, AL-3789 and AL-4940, were evaluated for glucocorticoid-mediated antiinflammatory activity in the in vitro U937 cell model of LPS-induced IL-1 induction and found to be devoid of glucocorticoid activity. Angiostatic steroids which lack glucocorticoid activity should be attractive drug candidates for treating ocular neovascular disease.

A current appreciation of sites for pharmacological intervention in allergic conjunctivitis: effects of new topical ocular drugs.

Two important realizations about pathophysiological mechanisms involved in allergic conjunctivitis have led to novel drug discovery efforts and new topical ocular medications for prevention and treatment of this prevent allergic disease. The first of these, interspecies and intraspecies mast cell heterogeneity, was established in the mid-1980's by investigators working in the field of asthma. It is now appreciated that secretory responses as well as effects of pharmacological agents differ depending upon the mast cell population studied. Two types of human mast cells, the tryptase containing (T) and the tryptase/chymase containing (TC) mast cells, have been characterized in a variety of tissues. Significantly, Irani et al. (1) demonstrated by immunohistochemical staining that the mast cells present in conjunctival tissues from patients with allergic conjunctivitis were 100% TC. Functional responses of human conjunctival mast cells to a variety of secretagogues (2) were consistent with their classification as TC or connective tissue type mast cells. Importantly, the studies by Miller et al. mentioned above allowed the harvesting and preparation of human conjunctival mast cells for use in drug screening studies. Utilization of these cells has led to the identification of Patanol, the most effective human conjunctival mast cell stabilizer available for topical use in allergic conjunctivitis (3). These same studies demonstrated the lack of mast cell stabilizing activity for cromolyn and nedocromil in these connective tissue type, TC containing, human conjunctival mast cells. Similar lack of effect was noted with these drugs on human skin mast cell degranulation (4). The second important discovery in the area of allergic conjunctivitis has been the demonstration that conjunctival epithelial cells may contribute to the perpetuation of the allergic response. A report from Gamache et al. (5) identified cytokines produced by human conjunctival epithelial cells following treatment with a number of stimuli. Significantly, Sharif et al. (6) subsequently identified functional histamine H1 receptors on these same cell types. Recently, Weimer et al. (7) have shown that exposure of human conjunctival epithelial cells to histamine leads to the production of pro-inflammatory cytokines IL-6 and IL-8. Importantly, treatment of the epithelial cells with drugs that possess histamine H1 antagonist properties prevents cytokine production. It is noteworthy that first generation anti-histamines antazoline and pheniramine are not potent inhibitors of histamine-stimulated cytokine synthesis in intact epithelial cells, while newer anti-histamines Emadine and levocabastine are more potent. Surprisingly, Patanol was more potent as an inhibitor of histamine-stimulated cytokine production by the epithelial cells than would be predicted from its histamine H1 antagonist affinity. These inhibitory effects on conjunctival epithelial cell production of pro-inflammatory cytokines may contribute to enhanced clinical activity noted with these recently approved drugs.

Inhibition of histamine-induced human conjunctival epithelial cell responses by ocular allergy drugs.

OBJECTIVE: To evaluate the effects of topical ocular drugs with histamine H1-antagonist activity on histamine-stimulated phosphatidylinositol turnover and interleukin (IL) 6 and IL-8 secretion from human conjunctival epithelial cells. METHODS: Primary human conjunctival epithelial cell cultures were stimulated with histamine in the presence or absence of test drugs. Phosphatidylinositol turnover was quantified by ion exchange chromatography and cytokine content of supernatants by enzyme-linked immunosorbent assay. RESULTS: Antazoline hydrochloride, emedastine difumarate, levocabastine hydrochloride, olopatadine hydrochloride, and pheniramine maleate attenuated histamine-stimulated phosphatidylinositol turnover and IL-6 and IL-8 secretion. Emedastine was the most potent in ligand binding, phosphatidylinositol turnover, and IL-6 secretion, with dissociation constant and 50% inhibitory concentrations of 1-3 nmol/L. Olopatadine, antazoline, and pheniramine exhibited similar H1-binding affinities (32-39 nmol/L). However, olopatadine was approximately 10-fold more potent as an inhibitor of cytokine secretion (50% inhibitory concentration, 1.7-5.5 nmol/L) than predicted from binding data, while antazoline and pheniramine were far less potent (20- to 140-fold) in functional assays. Levocabastine (dissociation constant, 52.6 nmol/L) exhibited greater functional activity (50% inhibitory concentration, 8-25 nmol/L) than either antazoline or pheniramine. CONCLUSIONS: Histamine-stimulated phosphatidylinositol turnover and cytokine secretion by human conjunctival epithelial cells are attenuated by compounds with H1-antagonist activity. However, antihistaminic potency alone does not predict anti-inflammatory potential. Olopatadine, emedastine, and levocabastine were notably more potent than pheniramine and antazoline. CLINICAL RELEVANCE: Selected topical ocular drugs with antihistaminic activity may offer therapeutic advantages to patients with allergic conjunctivitis by inhibiting proinflammatory cytokine secretion from human conjunctival epithelial cells.

Pharmacological validation of a feline model of steroid-induced ocular hypertension.

OBJECTIVE: To validate pharmacologically the feline model of steroid-induced ocular hypertension. METHODS: Serial studies were conducted in domesticated adult female cats trained to accept topical ocular drug administration and pneumotonometry. To establish intraocular pressure (IOP) values for each study, measurements were performed at the same time of day for 6 consecutive days. Beginning on day 7, cats received either steroid or vehicle administered topically to both eyes three times a day for approximately 28 days. The IOP measurements were performed daily. RESULTS: After 5 to 7 days of treatment with 0.1% dexamethasone or 1.0% prednisolone acetate, IOP began to increase, reaching peak values within 2 weeks. These values were sustained throughout dosing but declined rapidly to baseline upon cessation of treatment. Maximum IOPs for the dexamethasone- and prednisolone-treated groups averaged 4.5 +/- 0.3 mm Hg (n = 12) greater than the mean IOP value obtained in vehicle-treated cats. Cats treated with 0.25% fluorometholone, 1.0% loteprednol etabonate, and 1.0% rimexolone exhibited increases of 0.6, 1.2, and 1.7 mm Hg, respectively. These values were significantly lower than those observed following treatment with dexamethasone or prednisolone. CONCLUSIONS: The ocular hypertensive effects of selected anti-inflammatory topical ocular steroids in this model are consistent with clinical findings. CLINICAL RELEVANCE: This feline model is a useful tool for assessing the potential IOP liability of novel anti-inflammatory steroids.

Improved myeloperoxidase assay for quantitation of neutrophil influx in a rat model of endotoxin-induced uveitis.

Previously described models of endotoxin-induced uveitis quantify neutrophil influx into the eye using biochemical or direct cell count methods that result in an underestimation of ocular leukocyte accumulation following the inflammatory stimulus. We have optimized the rat model of endotoxin-induced uveitis by first overcoming interference in the biochemical assay of myeloperoxidase due to endogenous ocular reductants and cellular constituents containing free thiol functional groups. This was accomplished by simultaneously 1) extensively diluting soluble, interfering substances and 2) blocking tissue sulfhydril functional groups during tissue homogenization. Uveitis was induced in rats by subplantar injection of endotoxin. Twenty-four hours later, eyes were enucleated, homogenized, fractionated, and myeloperoxidase activity of neutrophils sedimenting with the membranous pellet was extracted. Previously published extraction procedures yielded only 40% of total assayable myeloperoxidase activity. Optimal recovery of myeloperoxidase activity (>twofold increase) was achieved only with two sequential extractions using 50 mM phosphate buffer (pH 7.4) containing 10 mM N-ethylmaleimide, and subsequent solubilization of myeloperoxidase activity by extraction with 0.5% hexadecyltrimethylammonium bromide in 50 mM phosphate buffer (pH 6.0). This modified extraction procedure and optimized myeloperoxidase assay conditions (300 microM hydrogen peroxide and 1.5 mM o-dianisidine) were then used to enhance the uveitis model. Maximum ocular neutrophil accumulation was observed at endotoxin doses of 100-200 microg. Total ocular neutrophil infiltrations ranged from 250,000 to 800,000 cells/globe. This leukocyte influx was inhibited dose-dependently by topical ocular administration of dexamethasone, with half-maximal inhibition observed at a concentration of 0.01%, w/v. Further validated by the correlation of biochemical results with histological evaluation, the refined methodology described in this report has application in assessing the ophthalmic therapeutic potential of antiinflammatory agents.

Histamine-stimulated cytokine secretion from human conjunctival epithelial cells: inhibition by the histamine H1 antagonist emedastine.

The present studies demonstrate that histamine induces the secretion of IL-6, IL-8 and GM-CSF from human conjunctival epithelial cells in a dose- and time-dependent manner. The histamine antagonists emedastine (H1), ranitidine (H2) and thioperamide (H3) were evaluated for their ability to inhibit secretion of these cytokines. Emedastine potently inhibited histamine-induced IL-6, IL-8 and GM-CSF secretion with mean IC50 values of 2.23, 3.42 and 1.50 nM, respectively. Ranitidine and thioperamide failed to inhibit cytokine secretion over a wide dose range. These data suggest that mast cell derived histamine may stimulate inflammatory cytokine production in allergic conjunctivitis via activation of epithelial cell H1 receptors. The histamine H1 antagonist emedastine potently inhibits this response.

Transient loss of prostaglandin synthetic capacity in rabbit iris-ciliary body following anterior chamber paracentesis.

The trauma-induced acute ocular inflammatory response has been characterized by investigating the kinetics of blood-aqueous barrier (BAB) breakdown, prostaglandin (PG) accumulation in the aqueous humor, and cyclooxygenase (PGH synthase) activity of the iris-ciliary body (ICB) following paracentesis in the NZA rabbit. BAB breakdown was assessed by quantifying plasma protein extravasation into the anterior chamber. PGE2 and 6-keto-PGF(1alpha) concentrations in the aqueous humor were quantified by radioimmunoassay. The capacity of ICB tissue homogenates to generate eicosanoids from exogenously supplied [I-14C]-arachidonic acid was assessed radiometrically by HPLC. Paracentesis resulted in a rapid and dramatic increase in aqueous humor PGE2 concentrations. Within 10 minutes, PGE2 concentrations increased 937-fold, from 6.2+/-4.9 pg/ml to maximal concentrations of 5810+/-3829 pg/ml. PG synthesis was followed temporally by an increase in aqueous humor protein, with peak levels (53.1 mg/ml) achieved within 30 minutes post paracentesis. Both PGE2 and protein levels gradually declined to near baseline levels 48 hours after trauma. ICB homogenates from naive animals produced significant amounts of eicosanoids (total PG=2.95 nmol/ 10 min/100 mg tissue). HHT (12 hydroxy-heptadecatrienoic acid) was produced in the greatest quantity, followed by PGE2alpha, PGI2, and TXB2/ PGF2 . Notably, following paracentesis, eicosanoid synthesis by the isolated ICB was observed to diminish abruptly. Formation of all eicosanoids was uniformly reduced by approximately 40% five minutes following paracentesis, with an 81% decrease in synthetic activity at 15 minutes. Eicosanoid synthetic capacity was only restored to baseline 48 hours post paracentesis. These findings suggest that, following ocular trauma, temporal changes occur in ICB PG synthetic activity that may impact on the selection of an optimal dosing paradigm for efficacy testing of topically administered NSAIDs.

Secretory response of mast cells contained in monodispersed human choroidal preparations.

BACKGROUND: Mast cells have been identified in the choroid of numerous species including man. However, functional studies involving these human mast cells have not been reported. In the current studies, the secretory response of human choroidal mast cells to various stimuli was examined using monodispersed choroidal cell preparations. METHODS: Monodispersed cell suspensions of human choroid were prepared from eye bank globes and the number, histamine content, and secretory response of mast cells in these preparations were determined. Choroids from 27 donors were used for these experiments. RESULTS: Cell suspensions contained an average of 15% mast cells. Mast cells stained positively with toluidine blue and exhibited the classical granular appearance upon electron microscopy. The amount of histamine contained in each mast cell was calculated to be 2.74+/-0.17 pg. Significant histamine release was observed following treatment with anti-human IgE, calcium ionophore A23187, concanavalin A, compound 48/80 and morphine. CONCLUSION: A method has been developed for obtaining monodispersed human choroidal mast cell preparations. The cells were functional as evidenced by their ability to release histamine upon immunological and nonimmunological stimulation. The degranulation noted following compound 48/80 and morphine challenge suggests that these human choroidal mast cells are analogous to connective tissue or chymase/tryptase-positive mast cells.

Secretion of proinflammatory cytokines by human conjunctival epithelial cells.

The production of cytokines by human conjunctival epithelial cells following stimulation was investigated. Primary cultures of human conjunctival epithelial cells were characterized by morphology and keratin expression. Cultured epithelial cells were treated with varying concentrations of lipopolysaccharide, interleukin (IL)-1 beta, calcium ionophore A23187, or phorbol myristate acetate, and cytokine secretion was determined over specified intervals. Culture supernatants and cell lysates were analyzed by ELISA for IL-1 beta, IL-3, IL-4, IL-5, IL-6, IL-8, IL-11, IL-1 receptor antagonist (IL-1ra), tumor necrosis factor-alpha (TNF-alpha), and granulocyte-macrophage colony stimulating factor (GM-CSF). With the exception of IL-1ra, unstimulated conjunctival epithelial cells produced cytokines at relatively low or undetectable levels. IL-1ra was detected in both culture supernatants and cell lysates under basal conditions. In response to stimuli, conjunctival epithelial cells secreted the proinflammatory cytokines TNF-alpha, IL-6, IL-8, and GM-CSF in a dose- and time-dependent fashion. After stimulation, the intracellular levels of IL-1ra increased in these cells but the supernatant-associated levels remained unchanged. None of the other cytokines evaluated (IL-1 beta, IL-3, IL-4, IL-5, and IL-11) were detected in supernatants or lysates of resting or stimulated cells. These findings suggest that conjunctival epithelial cells may contribute to the pathogenesis of human ocular diseases by production of proinflammatory cytokines. Further evaluation of these cells as targets of therapy is warranted.

Comparative effects of topical ocular anti-allergy drugs on human conjunctival mast cells.

BACKGROUND: The concept of mast cell heterogeneity is well established. Recent data indicate that human conjunctival tissue mast cells and human connective tissue mast cells respond to various secretagogues in similar fashion. It is now recognized that different mast cell populations respond differently to anti-allergic drugs. OBJECTIVE: The purpose of the study is to compare the effects of three new ocular anti-allergic drugs (nedocromil, olopatadine, and pemirolast) on mediator release from the target human conjunctival mast cell population with those of cromolyn sodium. The affinity of the compounds for the histamine H1 receptor was also compared. METHODS: A monodispersed suspension of partially purified human conjunctival mast cells was prepared from cadaver conjunctival tissue. Mast cells (5 x 10(3)) were challenged with anti-human IgE in the presence or absence of test drugs, and histamine content of the cell supernatants was determined using a specific radioimmunoassay. H1 receptor binding activity was assessed using a radioligand binding assay. RESULTS: Cromolyn and pemirolast (100 nM to 1 mM) failed to significantly inhibit histamine release from human conjunctival mast cells using exposure times of 1 and 15 minutes prior to challenge. Using identical nedocromil concentrations and exposure times, statistically significant (P < .05) inhibition (28%) of histamine release was observed at only the 100 microM concentration and 1-minute exposure time. In contrast, olopatadine inhibited histamine release in a concentration-dependent fashion (r = 0.891, n = 59, IC50 = 653 microM). Only olopatadine exhibited significant H1 receptor binding activity at relevant concentrations (Ki = 36 nM, n = 13). CONCLUSIONS: These data indicate that olopatadine possesses anti-allergic activity in the appropriate targets for topical ocular anti-allergic drug therapy, human conjunctival mast cells. Coupled with the compound's antihistaminic activity, this suggests that olopatadine will have efficacy advantages in allergic conjunctivitis patients over the other drugs tested.

Characterization of the ocular antiallergic and antihistaminic effects of olopatadine (AL-4943A), a novel drug for treating ocular allergic diseases.

Olopatadine (AL-4943A; KW-4679) [(Z)-11-[3-(dimethylamino)propylidene]-6, 11-dihydrodibenz[b,e]oxepine-2-acetic acid hydrochloride] is an antiallergic/antihistaminic drug under development for topical ocular use. The effects of the compound on release of proinflammatory mediators (histamine, tryptase and prostaglandin D2) from monodispersed human conjunctival mast cells were assessed. Histamine receptor subtype binding affinities and functional potencies were determined with ligand binding and phosphoinositide turnover assays, respectively. Olopatadine inhibited the release of histamine, tryptase and prostaglandin D2, in a concentration-dependent manner (IC50 = 559 microM). Evaluation of the interaction of olopatadine with histamine receptors revealed a relatively high affinity for the H1 receptor (Ki = 31.6 nM, pKi = 7.5 +/- 0.1, n = 7) but lower affinities for H2 receptors (Ki = 100 microM, pKi = 4.0 +/- 0.19, n = 7) and H3 receptors (Ki = 79.4 microM, pKi = 4.1 +/- 0.16, n = 7). The H1 selectivity of olopatadine was superior to that of other ocularly used antihistamines studied, such as ketotifen, levocabastine, antazoline and pheniramine. The profiling of olopatadine in 42 nonhistamine receptor binding assays revealed that olopatadine interacts with only two nonhistamine receptor/uptake sites to any significant degree (pIC50 < or = 5-6). Olopatadine inhibited histamine-induced phosphoinositide turnover in human conjunctival epithelial cells (IC50 = 10 nM, pIC50 = 8.0, n = 4) and in other human ocular cells (IC50 = 15.8-31.6 nM, pIC50 = 7.5-7.8) and exhibited apparent noncompetitive antagonist properties in these cells, with an estimated dissociation constant (Kb) of 19.9 nM (pKb = 7.7, n = 6). This combination of mast cell mediator release inhibition and selective H1 receptor antagonism suggests that olopatadine may be particularly useful in the treatment of ocular allergic diseases. Indeed, olopatadine has recently shown clinical efficacy in an allergic conjunctivitis model in human subjects.

The in vitro and in vivo ocular pharmacology of olopatadine (AL-4943A), an effective anti-allergic/antihistaminic agent.

Olopatadine (AL-4943A; KW-4679) [(z)-11-[3-(dimethylamino)propylidene]-6, 11-dihydrodibenz[b,e]oxepine-2 acetic acid hydrochloride] is an anti-allergic agent which inhibits mast cell mediator release and possesses histamine H1 receptor antagonist activity. Studies were conducted to characterize the in vitro and in vivo pharmacological profile of this drug relevant to its topical ocular use. AL-4943A inhibits histamine release in a concentration-dependent fashion (IC50 = 559 microM) from human conjunctival mast cell preparations in vitro. Histamine release was not stimulated by AL-4943A at concentrations as high as 10 mM. In contrast, ketotifen stimulated histamine release at concentrations slightly higher than effective inhibitory concentrations. AL-4943A did not display any in vitro cyclooxygenase or 5-lipoxygenase inhibition. Topical ocular application of AL-4943A effectively inhibits antigen- and histamine-stimulated conjunctivitis in guinea pigs. Passive anaphylaxis in guinea pig conjunctiva was attenuated by AL-4943A applied 30 min prior to intravenous or topical ocular antigen challenge (ED50 values 0.0067% and 0.0170%, w/v, respectively). Antihistaminic activity in vivo was demonstrated using a model of histamine-induced vascular permeability in guinea pig conjunctiva. AL-4943A applied topically from 5 min to 24 hrs prior to histamine challenge effectively and concentration-dependently inhibited the vascular permeability response, indicating the compound has an acceptable onset and a long duration of action. Drug concentrations 5-fold greater than those effective against histamine-stimulated conjunctival responses failed to inhibit vascular permeability responses induced with either serotonin or Platelet-Activating-Factor. These data indicate that the anti-histaminic effect observed with AL-4943A is specific. These anti-allergic/antihistaminic activities of AL-4943A observed in preclinical model systems have been confirmed in clinical trials in allergic patients.